1. Field of the Invention
The present invention relates generally to the field of diagnostic medicine, most particularly, to the field of methods for diagnosing organ graft rejection. The invention relates to methods for monitoring and detecting organ rejection in a patient through immunodiagnostic assays which measure free secretory component (FSC) in a biological sample.
In that particular embodiments of the present invention relate to the measurement of FSC in a urine sample, the field of the present invention also relates to urine sample analysis for the detection of organ rejection. In most preferred embodiments of the described invention, a method for monitoring and detecting allograft rejection is provided through the monitoring of urine concentrations of the glycoprotein free secretory component. The described invention most particularly relates to the field of methods for detecting renal allograft rejection, as the procedures disclosed herein are capable of distinguishing renal allograft rejection from other forms of renal and extra renal problems in kidney transplant patients.
The present invention also relates to the field of diagnostic assay kits, as a diagnostic kit for use in the screening of kidney transplant patients to detect early kidney rejection is provided.
2. Description of the Related Art
Approximately 10,000 kidney transplants are performed annually in the United States. Despite the availability of potent immunosuppressive agents, graft rejection remains the main complication of renal transplantation. For example, approximately 50% of all renal allograft recipients are thought to suffer at least one episode of graft rejection. The likelihood of kidney loss due to rejection is highest during the first year post transplant (10-20%), but a small proportion (3-5%) of kidneys are rejected each year even after that.sup.1.
The statistics indicate that graft rejection is often not detected early enough in the rejection episode to allow initiation of countervailing treatment, which would effectively halt the rejection process, in time to prevent the organ rejection. A method for the early detection of graft rejection would thus be an important clinical tool for maintaining the viability of a transplanted organ through early detection in the rejection process. The clinician would thus be provided an opportunity to administer immunosuppressive agents to the patient at a time when the rejection process could be effectively halted and/or prevented altogether.
The diagnosis of acute allograft rejection is classically based on the presence of one or more symptoms. For example, symptoms of acute allograft rejection include weight gain, reduced urine output, increased serum creatine concentrations, hypertension, fever, and graft enlargement and tenderness. However, the use of these symptoms alone to detect rejection is not adequate. Currently, most transplant rejection episodes are detected by measuring periodically the function of the transplanted kidney, for example by using biochemical tests such as assays which measure serum creatine concentrations alone.
Other non-invasive methods for detecting renal graft rejection are under investigation, and depend primarily on the measurement of "indicators" of activation of the immune system. For example, the quantification of T-lymphocyte subsets.sup.2 or soluble T-cell surface receptors (e.g., interleukin 2 receptor).sup.3 in the blood have been used to monitor graft rejection.
Unfortunately, other complications of the transplant procedure, including urinary tract infection, urinary obstruction and toxicity of the immunosuppressive therapy itself (particularly with cyclosporin A), often mimic the clinical and/or laboratory findings of graft rejection. For example, kidney toxicity to cyclosporin A (a drug used to prevent rejection of kidneys) often results in elevated levels of creatinine in the serum and adenosine deaminase binding protein in urine. The measurement of these constituents would therefore fail to distinguish kidney rejection per se from toxicity to the immunosuppressive agents used to prevent rejection. Such would render false positive results for graft rejection in a patient. In addition, other unrelated illnesses can produce clinical and laboratory findings which resemble those of renal transplant rejection. For instance, T-cell activation may occur during common vital infections as well as during episodes of renal allograft rejection.sup.4, again lending to the occurrence of false positive laboratory results in a patient.
In addition, many abnormalities of the kidney are reflected in changes in the volume and composition of the urine. Therefore, urine tests might provide a more direct and therefore specific, indication of renal allograft rejection than assays for components included in the blood. Measurement of renal tubular and serum proteins in the urine, including .beta.-2 microglobulin.sup.5,6, gamma glutamyl trans-peptidase.sup.6, N-acetyl-.beta.-D-glucosaminidase.sup.7,8, adenosine deaminase-binding protein.sup.10,11 (produced by proximal nephron), complement components, and lysozyme.sup.4,9, have all been proposed. However, none of these markers have been shown to distinguish the injury from renal allograft rejection apart from other renal injuries.
Presently, renal biopsy remains the most definitive test to specifically diagnose renal allograft rejection. However, this method has major limitations. For example, since the biopsy procedure itself has complications, and since a portion of the renal transplant is removed during each biopsy, transplant biopsy cannot be performed on a routine or even frequent basis to monitor renal allograft rejection. In addition, the invasive nature of a renal biopsy is both uncomfortable and inconvenient for patient subjects. Accurate interpretation of the renal transplant biopsy also demands the expertise of a pathologist with extensive experience in analyzing a biopsy sample for evidence of renal transplant rejection. Hence, renal biopsies are reserved for those patients that demonstrate other clinical and/or laboratory evidence of renal allograft rejection limiting its use or potential use in detecting early graft rejection.
While no direct evidence for the induction of secretory component in kidney cells by injury is known to exist, one immunohistologic study of renal autopsy tissue demonstrated that secretory component was much more prominent in the distal tubules when renal disease was present.sup.28. The present inventors have recently examined a representative number of biopsies from renal allografts from patients undergoing graft rejection, and compared these to biopsies from patients with cyclosporin toxicity, using immunofluorescence assays with monoclonal antibodies to secretory component. From these preliminary observations, the inventors found that kidneys undergoing rejection expressed much more secretory component in their distal tubules compared to kidneys from patients with cyclosporin toxicity (unpublished data). The inventors therefore propose a never-before-described method for discerning kidney rejection in patients receiving immuno-suppressive agents such as CSA, by monitoring urinary free secretory component. Concentrations of FSC are not observed to increase with the administration of immunosuppressive drugs (or reactions thereto), thereby essentially eliminating false positive results for kidney rejection.
Secretory component is an 80,000 molecular weight fragment of an integral (trans) membrane glycoprotein, termed "polymeric immunoglobulin receptor" (PIgR). PIgR is synthesized by secretory epithelial cells which line the mucosal surfaces of the body.sup.12,13. It mediates binding, endocytosis and vectorial transport of polymeric IgA (pIgA), and to a lesser extent IgM.sup.14, from the basal lateral to the apical surface of the epithelial cell.sup.15-19. Here, the PIgR is proteolytically cleaved near the outside surface of the epithelial cell.sup.20, releasing the N-terminal segment, termed secretory component (SC).
The secreted secretory component, complexed with pIgA, is termed "secretory IgA" (sIgA).sup.12. In this complex, secretory component is thought to protect the IgA from proteolytic cleavage. The large amounts of sIgA found in saliva and gastrointestinal secretions, lung secretions, cervicovaginal secretions, breast milk, and tears are thought to constitute the first line of immunological defense against some mucosal infections. The smaller amount of sIgA found in urine may also help protect against urinary tract infection.sup.22.
When secretory component is secreted from the epithelial cell without any immunoglobulin attached, it is termed free-secretory component (FSC). FSC has also been reported in all external secretions in smaller amounts.sup.23. The exact function of FSC remains unknown. It is hypothesized that the synthesis of secretory component in some tissues may be regulated by hormones.sup.24, and other soluble mediators, such as cytokines involved in inflammation. In cultured colon carcinoma cells, the cytokines interferon gama and tumor necrosis-.alpha. and interleukin-4, alone and in combination, have been shown to increase the production and release of secretory component..sup.25-27
The term "secretory component", as used in describing the present invention, refers to either the conjugated (sIgA-associated) or unconjugated (FSC) form of the secretory component molecule. Free secretory component (FSC), as used in describing the present invention, refers only to the free, unconjugated form of the secretory component molecule.
Since secretory component is normally secreted from the luminal surface of epithelial cells, the inventors hypothesize that it is likely that increased production of secretory component in the kidney will increase excretion of sIgA and/or secretory component in the urine. A recent study quantified sIgA by ELISA in the urine of patients with a variety of renal disorders.sup.29. Compared to healthy subjects, patients with IgA nephritis and other forms of chronic glomerulonephritis had elevated urinary sIgA. Urinary sIgA levels were only slightly increased in heavy proteinuria. Additionally, the highest concentrations of urinary sIgA (22-fold above normal) were found in patients with urinary tract infections. Renal allograft recipients with stable graft function had urinary sIgA levels that were only slightly increased above those of normal individuals. These results suggest that urinary sIgA production is enhanced by renal inflammation, perhaps due to induction of local secretory component and/or IgA production.
Urinary free secretory component (FSC) excretion, (unconjugated secretory component) has not been studied as extensively. As will be recalled, FSC (or free secretory component) is produced by the distal nephron. It has been observed that the level of expression and excretion of FSC may be increased by renal inflammatory reactions like infection.sup.28 or allograft rejection, as well as by tubular injury. One recent study demonstrated that urinary FSC did not differ between persons with acute/recurrent urinary tract infections, and normal controls..sup.32 Nor did urinary FSC levels differ between patients with IgA deficiency and age and sex--matched normal adult control subjects..sup.32
A major characteristic of the secretions that protect mucosal surfaces is that they contain products of the polymeric immunoglobulin transport system, namely secretory immunoglobulins (sIgA and sIgM) and free secretory component (SC). SC expression has even been demonstrated as present in fetal tissues. For example, by the seventh week of gestation, even before the fetus is able to produce Ig, SC has been detected in fetal tissues..sup.4 Free SC (free secretory component) has been observed by the inventors in human amniotic fluid. Based on reactivity with antisera to secretory component, the IgA of amniotic fluid is thought to be sIgA immunoglobulin containing secretory component.sup.33.
Amniotic fluid is known to serve as an immunological barrier between the fetus and maternal external environments, thereby protecting against intrauterine infections. As already noted, amniotic fluid has been reported to contain low levels of sIgA and other proteins typical of external secretions, but these proteins are not usually considered an immunologic secretion.sup.4. Thus, the role and sources of the various forms of secretory component (SC) in amniotic fluid have not yet been completely characterized.
The present inventors have found that the concentration of SC in amniotic fluid (AmF) varies with the gestational age of the fetus. Additionally, the inventors have found that most of the SC in human AmF exists in its free form (FSC). In this regard, the inventors observed that amniotic fluid from full term pregnancies contained FSC in concentrations comparable to other external secretions, such as saliva or bile.sup.42.
The fetal ufogenital system and the amniotic membrane have been demonstrated to contribute to FSC levels and sIgA levels in amniotic fluid. Given those observations already collected by the inventors and others regarding FSC and SC in fetal tissues and AmF, the inventors propose a method for monitoring fetal kidney abnormalities through monitoring fetal secretory component levels in amniotic fluid. For example, this proposed method may be used to monitor the disease known as Potter's Syndrome (a disorder characterized by abnormal fetal kidney development). Detection of kidney malfunction early in pregnancy by monitoring AmF levels of FSC would provide an extremely valuable diagnostic tool for following fetal kidney development. Once detected, the kidney-malfunctioning fetus might be treatable by the clinician and alert the patient to the condition.
A simple, non-invasive method for rapid detection of kidney and other organ rejection episodes would have the dual advantage of requiring no venipuncture or biopsy and further allowing testing at virtually any clinic or office remote from actual transplantation medical centers. More specifically, a urinary FSC test to monitor kidney rejection as a screening procedure would have practical utility as an initial non-invasive procedure that would quickly alert the physician of the need for further, more extensive and/or patient involved evaluation and treatment. Quantification of urinary FSC may also be useful in distinguishing inflammatory renal disease (rejection or infection) from renal toxicity precipitated from immunosuppressive drugs, such as cyclosporin A.
A simple, non-invasive diagnostic test which could serve as an initial screening procedure for detecting renal transplant rejection, or could distinguish rejection from other diseases that produce the same symptoms and biochemical changes, could improve greatly the prognosis of patient kidney transplant recipients. As allograft rejection can often be treated effectively if detected early, the availability of an effective screening test for early graft rejection detection which could be performed conveniently on a relatively frequent periodic basis would potentially improve the survival of both the transplanted organ, and the transplant patient. Ultimately, such a test could also reduce the high cost of treating chronic renal failure.